Structural basis of ligand binding modes of human EAAT2

In the central nervous system (CNS), excitatory amino acid transporters (EAATs) mediate the uptake of excitatory neurotransmitter glutamate and maintain its low concentrations in the synaptic cleft for avoiding neuronal cytotoxicity. Dysfunction of EAATs can lead to many psychiatric diseases. Here we report cryo-EM structures of human EAAT2 in an inward-facing conformation, in the presence of substrate glutamate or selective inhibitor WAY-213613. The glutamate is coordinated by extensive hydrogen bonds and further stabilized by HP2. The inhibitor WAY-213613 occupies a similar binding pocket to that of the substrate glutamate. Upon association with the WAY-213613, the HP2 undergoes a substantial conformational change, and in turn stabilizes the inhibitor binding by forming hydrophobic interactions. Electrophysiological experiments elucidate that the unique S441 plays pivotal roles in the binding of hEAAT2 with glutamate or WAY-213613, and the I464-L467-V468 cluster acts as a key structural determinant for the selective inhibition of this transporter by WAY-213613.

however this reviewer questions the usefulness of EAAT2 inhibitors clinically given that deficiencies in EAAT2 cause disease. The authors point to Ref 11 for evidence that EAAT2 inhibitors may be clinically useful however this is actually not mentioned in the paper referred to at all.
2. While the structures provided by Zhang et al. provide significant insight as to the mechanism of inhibition by WAY-213613, for this paper to be complete, additional experiments are required that delineate a structure activity relationship. Functional experiments investigating the residues that coordinate WAY-213613 need to be conducted. For example, mutating these residues to the equivalent residues in other EAATs that have a lower affinity for WAY-213613 will help pinpoint exactly what residues are critical for the specific inhibition of EAAT2.
3. The cryoEM maps provided show clear density for glutamate and WAY-213613. Interestingly, they also show density for a DDM molecule in the glutamate binding site in the WAY-213613-bound structure. This reviewer feels that this is a very interesting observation that warrants discussion in the manuscript. Could it be that inhibition by WAY-213613 causes other molecules such as detergents -or more physiologically relevant -lipids to interact with residues in the glutamate binding site and also contributing to inhibition of transport?
4. This manuscript lacks any discussion of the results or a conclusion -these are essential aspects to scientific literature and without them this manuscript is incomplete. This reviewer feels especially strongly about this given that inhibition of glutamate transporters therapeutically is somewhat controversial and warrants, at the very least, further thought and discussion.
• Line 27: It should be noted that excessive concentrations are neurotoxic.
• Line 32: "Buffering glutamate" doesn't really make sense. Should be replaced with "transporting glutamates back up into the pre-synapse or astrocytes".
• Lines 39-40: As noted above in major comments, more detail is warranted to explain how EAAT2 may be an important therapeutic target.
• Line 41: Ph for GltPh should be in subscript.
• Line 47: The substrate binding site is described as being "close to HP1 and HP2". There are several substrate-bound structures of glutamate transporters and given that this is a key finding of this paper, what is already known about the substrate binding site should not be glazed over. More detail is required.
• Line 53: It would be useful at this point of the paper to know the affinities of WAY-213613 for different human EAATs rather than at line 157.
• Supplementary Figures 2 and 3: It is difficult to see the densities. Please change the colors. Also, the densities for substrates and inhibitors should be shown in these figures.
• Lines 67-68: References are missing for the structures referred to.
• Line 76: What do the authors mean by "HP1 is attached to the inner membrane"? This reviewer is not familiar with the concept of a protein attaching to the membrane.
• Supplementary Figure 4 and Lines 86-88: While it has been described that CHS-resembling densities could be identified, this is not clearly demonstrated in Supplementary Figure 4. The authors should show how CHS fits into the density with more detail if this is their claim.
• Supplementary Figure 4: The colors for the hairpins are only shown in one of three protomers. Either say this in the legend or show it in all three protomers. Also, HP2 is listed as being blue when really it looks purple. Furthermore, WAY-213613 is said to be colored in pink, when it is actually red, and this can be very confusing with the pink-magenta used to color HP2 in this panel. Please use more distinct and consistent colors for these figures. Labels may also be helpful.
• Lines 93-95: This sentence seems out of place and is unnecessary at this point in the manuscript.
• Figure 2: I see beta sheets in the structure, but no beta sheets have been mentioned. Please explain.
• Figure 2: Density for glutamate should be shown in a close-up view.
• Lines 101-107: How does this substrate binding site compare to other substrate-bound structures? This point warrants discussion and prior work should be acknowledged here.
• Line 109-110: "Upon the glutamate binding" should be replaced with "when glutamate is bound" because here, we are talking about the release of substrate.
• Lines 115-118: Is S441 important for substrate transport? This warrants functional investigation, especially since it is not conserved amongst the different homologues. Why are these interactions of S441 significant?
• Lines 124-125: "WAY-213613 has no effect on ionotropic and metabotropic glutamate receptors and thus it's a potential tool for elucidating the function of hEAAT2." We already know the function of EAAT2; this is a vague sentence and does not tell the reader why they should care about understanding how WAY-213613 binds to EAAT2. Careful thought, consideration and explanation is warranted here.
• Line 128 and Figure 3B: A more zoomed-in figure of WAY-213613 in the density map should be shown.
• Figures 2D and 3D are mirror images of each other. Please amend for ease of interpretation.
• In several figures, two structures are overlaid with one structure shown in gray and the other shown in green as detailed by the figure legend. In these figures, HP2 is colored in red, but it is not detailed in the figure legend which structure this red (HP2) belongs to. Please clarify.
• Supplementary Figure 6: Please put PDB IDs into figure legends.
• Lines 168-181: There are also structures of TBOA bound to GltPh in outward-facing structures. These should also be discussed; how do they fit into this story?
Reviewer #3 (Remarks to the Author): EAATS play a critical role in maintaining low glutamate concentrations in the synaptic cleft of the CNS and they are involved in several neurological diseases as well as cancer. Therefore, it is important to understand how these proteins work and how different ligands and inhibitors bind to these transporters. A few structures of the glutamate transporter family have been already published, and the complete transport mechanism of the SLC1A family is well known.
The authors describe 2 new Cryo-EM structures of EAAT2 in the presence of substrate and the inhibitor WAY-213613 and they reveal a new inhibitor binding site for this glutamate transporter family. The manuscript is well written and the methodology used is appropriate.
Although the authors present the structure of the human EAAT2 in the presence of glutamate and the inhibitor WAY-213613, no other experiment was performed to understand and validate the inhibition mechanism by WAY-213613. No mutagenesis analysis or functional analysis was done to corroborate the findings in the Cryo-EM structures. There is no validation in the paper, other than the structures, suggesting that this new inhibitor binding site is real. This could be done, for example, by mutating key amino acids that interact with the inhibitor WAY-213613 to see if the inhibition could be abolished. This could be analysed either by binding experiments or structural determination. This would be a good way to validate the new inhibitor binding site.
I believe the validation reports are mixed up at my end. In the validation report that seems to correspond to the EEAT2-WAY213613, the structures of the ligands Y01 have issues in bond lengths and bond angles. As the paper's main outcome is a new inhibitor binding site of the EAATs, it would be good to see omit maps for the ligands to better appreciate the densities around them.
Another aspect that could be investigated is the difference between monomers. Have the authors observed the same density for the WAY-213613 in the three monomers forming the trimer? A symmetry expansion and focused refinement could be performed to identify any structural variation between protomers.

Reviewer #1 (Remarks to the Author):
Zhang et. al. report human EAAT2 structures determined in glutamate and inhibitors bound states. These new structures are important, but the manuscript can be considerably improved with functional data to validate binding sites for glutamate and the inhibitor.

Reply:
We appreciate reviewer's suggestions for the improvement of our manuscript. We have performed electrophysiological experiments with wild-type hEAAT2 and mutants transiently transfected HEK293T cells to validate the binding sites for the substrate glutamate and the inhibitor WAY-213613.
We supplemented functional characterization of hEAAT2 in revised manuscript. In the lines 75-87 it reads "In addition to mediating excitatory amino acid transport, hEAAT2 also acts as an anion-selective channel [27][28][29] . The hEAAT2-associated current includes three components: glutamate-induced anion current, Na + -dependent anion leak current and glutamate transport current 30,31 . The competitive inhibitors can block all three components of the current 32 . To gain more insights into glutamate and inhibitor binding sites, we carried out electrophysiological experiments using whole cell patch-clamp technique under similar conditions to the ones described previously 25 . In line with previous reports 31,33,25 , application of substrate glutamate increased the amplitudes of anion leak currents ( Supplementary Fig. 1a). In contrast, the WAY-213613 was able to block the anion leak conductance ( Supplementary Fig. 1b). The activation of anion leak currents mediated by glutamate and inhibition of the ones by WAY-213613 were both dose dependent and could be fitted into a Michaelis-Menten-type equation, yielding a Km of 30±2.5 μM for glutamate and an apparent Ki of 0.07±0.03 μM for WAY-213613 ( Fig.  1a-1b), both of which are consistent with previous reports 34,26 .".
We supplemented a short discussion about the functional analysis for the residues D475 and R478 in revised manuscript. In the lines 142-153 it reads "To validate the glutamate binding site, we mutated D475 TM8 and R478 TM8 to alanine separately (D475A TM8 and R478A TM8 ). Electrophysiological experiments showed that glutamate failed to activate anion currents of these two mutants at the concentration of glutamate up to 1 mM, as compared with that of wild-type hEAAT2 (Fig. 2e). To rule out the possibility that mutation in either residue might disrupt the activity of hEAAT2 as an anion channel, we also tested the inhibitory effects of WAY-213613 on anion currents of these two mutants. As a result, both mutants, D475A TM8 and R478A TM8 , still could mediate an inward anion current, evidenced by an apparent outward current induced by the WAY-213613, although the apparent Ki of WAY-213613 was reduced to 30.48 μM and 1.68 μM for D475A TM8 and R478A TM8 , respectively (Fig. 2f). Therefore, we speculate that the residues D475 TM8 and R478 TM8 are critical for the binding of hEAAT2 with glutamate and might also participate in the binding of this transporter with WAY-213613.".
Similarly, we supplemented a short discussion about the functional analysis for the residue S441 in revised manuscript. In the lines 165-178, it reads "To investigate the functional role of the S441 HP2 , we substituted this residue with glycine (S441G HP2 ) and performed electrophysiological recordings by applications of various concentrations of glutamate. Strikingly, we found that the S441G HP2 mutant displays higher sensitivity to glutamate with the Km at 0.40±0.03 μM (Fig. 2h), which is increased by ~77-fold higher than that of wild-type hEAAT2, although the S441 HP2 is not directly involved in the glutamate binding ( Fig. 2c and 2d). This residue is exclusively present in the hEAAT2, as compared with the conserved glycine at the corresponding position in other homologs ( Supplementary Fig. 7). We speculate that the unique residue S441 HP2 probably provides additional interactions to stabilize the HP2 at a sealed conformation and prohibit glutamate release from the intracellular side. Thus, mutation in the S441 HP2 might rupture these interactions, facilitate the glutamate release, accelerate the glutamate uptake cycle, and consequently biases the hEAAT2 towards a channel with higher open probability. The apparent Ki of WAY-213613 for the mutant S441G HP2 was reduced to 0.26 μM (Fig.  2f), which hints that this site also affects the binding of the inhibitor in a certain way.". μM glutamate and presented after Na + dependent leak current was subtracted. Sample sizes (n) tested for wild-type hEAAT2, D475A TM8 or R478A TM8 are 5, 4, 5 cells. f. WAY-213613 dose-response relationships for the D475A TM8 , R478A TM8 or S441G HP2 mediated currents. WAY-213613 was varied at the following concentrations: 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM for D475A TM8 and R478A TM8 ; 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM for S441G HP2 . Currents were normalized to the maximal current recorded after application of 30 μM or 100 μM WAY-213613. Sample sizes (n) tested from low to high concentrations are listed as follows: n=4, 5, 5, 5, 5, 9 cells for D475A TM8 ; n=5, 5, 5, 4, 5, 12 cells for R478A TM8 ; n=4, 4, 4, 5, 5, 4, 7 cells for S441G HP2 . The lines represent the best fits to a Michaelis-Menten-like equation. The data of wild-type hEAAT2 from tested from low to high concentrations are 4, 5, 5, 5, 12, 5 cells.
Also, we supplemented a short discussion about the functional analysis for the residues I464, L467, and V468 in revised manuscript. In the lines 237-245, it reads "To validate the above hypotheses, we designed three mutations, including I464V TM8 , L467I TM8 and V468I TM8 . The electrophysiological experiments indicate that mutations do not affect glutamate binding. For these three mutants, application of glutamate in the external solution can significantly activate anion current with similar efficacy as that of wild-type hEAAT2 (Fig. 4d). However, these mutations lead to a significant decrease in sensitivity to WAY-213613, with Ki increased to ~0.17 μM, ~0.46 μM and ~0.20 μΜ for I464V TM8 , L467I TM8 and V468I TM8 mutants, respectively (Fig. 4e), supporting our speculations that the I464 TM8 , L464 TM8 and V468 TM8 residues are crucial for the binding specificity of hEAAT2 with WAY-213613.". tested from low to high concentrations are listed as follows: n=5, 5, 5, 5, 5, 5 cells for the mutant I464V TM8 ; n=4, 4, 5, 5, 5, 5 cells for the mutant L467I TM8 ; n=5, 5, 5, 5, 4, 6 cells for the mutant V468I TM8 . e. WAY-213613 dose-response relationships for the I464V TM8 , L467I TM8 or V468I TM8 mediated currents. Minor comments: 1. The manuscript appears to be cursorily written and can be improved. For example, in line 50, hASCT1 and hASCT2 do not transport glutamate.
2. The authors may also want to compare EAAT2/glutamate with other substrate bound EAAT structures.
Reply ： We thank reviewer's suggestion. We compared the structures between the outward-facing hEAAT1 Asp and the inward-facing hEAAT2 Glu in the lines 123-129 of the main text. It now reads "Using the scaffold domain as a reference, we performed structural comparison between the outward-facing hEAAT1 Asp (PDB ID: 5LLU) 19 and the hEAAT2 Glu structures and found that the transport domain of hEAAT2 Glu structure is displaced by ~17 Å across the membrane towards cytoplasmic side relative to that of hEAAT1 Asp , suggesting that the hEAAT2 Glu structure is determined in an inward-facing conformation ( Supplementary Fig. 5). Such conformational change of the transport domain is also consistent with previous observations 24 .".
We supplemented a supplementary Fig  We also have compared the structures between hEAAT2 and eukaryotic homologs (hEAAT1, hEAAT3, hASCT2) and prokaryotic homologs (GltPh, GltTk) in the lines 136-142 of the main text. It now reads "By comparison with eukaryotic homologs (hEAAT1 19 , hEAAT3 20 , hASCT2 22 ) and prokaryotic homologs (GltPh 14 , GltTk 17 ), key residues involved in substrate binding are highly conserved between hEAAT2 and the above-mentioned homologs ( Supplementary Fig. 6). However, R478 TM8 in the hEAAT2 is substituted by C467 TM8 at the corresponding position in the neutral amino acid transporter hASCT2 ( Supplementary Fig. 6e), which contributes to the substrate selectivity of hASCT2 for neutral amino acids 20 .".  Structural comparison of the transport domains between hEAAT2 Glu (pale green) and hEAAT3 Asp (PDB ID: 6X2Z, wheat). c. Structural comparison of the transport domains between hEAAT2 Glu (pale green) and GltPh Asp (PDB ID: 6X15, white). d. Structural comparison of the transport domains between hEAAT2 Glu (pale green) and GltTk Asp (PDB ID: 6R7R light pink). e. Structural comparison of the transport domains between hEAAT2 Glu (pale green) and hASCT2 Asp (PDB ID: 6GCT, salmon). In Supplementary   Fig. 6a-e, the HP1 tip of hEAAT2 Glu is highlighted in blue to be differentiated from those of other homologs.
3. Since both EAAT/glutamate and EAAT/Asp structures are available, can these structures explain why EAAT prefers Glu over Asp (or lack of selectivity). Reply: We appreciate reviewer's comment. Previous reports showed that the Km values of hEAAT1, hEAAT2, and hEAAT3 for glutamate were 48 μM, 97 μM and 62 μM, respectively; the Km values of hEAAT1, hEAAT2 and hEAAT3 for aspartate were 60 μM, 54 μM and 47 μM, respectively (PMID: 7521911). Therefore, the hEAATs do not exhibit a significant difference in substrate selectivity between glutamate and aspartate. Our structural comparison also supports these biochemical experimental results, since the key residues involved in substrate binding are highly conserved (Figure 4*). Figure 3d, Leu447 should be Leu467?

In
Reply: Thank you very much for the reminder. We have updated the Fig. 3d and added the residue Leu467 in the revised Fig. 3d. Moreover, we also adjusted the orientation of the WAY-213613. The new orientation is similar to the one in the Fig. 3c.

Reviewer #2 (Remarks to the Author):
SUMMARY In this manuscript, Zhang et al. solve the structures of human EAAT2 in complex with its substrate glutamate and inhibitor called WAY-213613 using cryo-EM. The good resolution of these structures allows for both the substrate and inhibitor to be placed in the map and consequently the mechanism of inhibition by WAY-213613 to be delineated as competitive antagonism. Reply: We appreciate reviewer's positive comments and suggestions for improving our manuscript. We have made revisions as described below.

MAJOR COMMENTS
1. It is mentioned that inhibitors of EAAT2 may be an important therapeutic target, but the discussion needs more depth. It seems that the authors are claiming that inhibition of EAAT2 may be clinically however this reviewer questions the usefulness of EAAT2 inhibitors clinically given that deficiencies in EAAT2 cause disease. The authors point to Ref 11 for evidence that EAAT2 inhibitors may be clinically useful however this is actually not mentioned in the paper referred to at all.

Reply:
Thank you for pointing out this confusing statement. We factually want to express that hEAAT2, but not inhibitors of hEAAT2, is an important therapeutic target. We have clarified this in the lines 45-48 of revised manuscript. Now it reads "Deficiency of hEAAT2 causes progressive neuronal death, and psychiatric or neurological diseases, including major depressive disorder, epilepsy, Alzheimer's disease, stroke, Parkinson's disease and amyotrophic lateral sclerosis (ALS), and thus hEAAT2 represents a potential therapeutic target 6-8 .".
2. While the structures provided by Zhang et al. provide significant insight as to the mechanism of inhibition by WAY-213613, for this paper to be complete, additional experiments are required that delineate a structure activity relationship. Functional experiments investigating the residues that coordinate WAY-213613 need to be conducted. For example, mutating these residues to the equivalent residues in other EAATs that have a lower affinity for WAY-213613 will help pinpoint exactly what residues are critical for the specific inhibition of EAAT2.

Reply:
We appreciate reviewer's constructive suggestion about the functional experiments. We have performed electrophysiological experiments for wild-type hEAAT2 and mutants to validate these key residues coordinating with WAY-213613. It turns out that the I464, L467, and V468 residues from TM8 play pivotal roles in the binding hEAAT2 with WAY-213613. We have added a brief discussion about the functional analysis in the lines 237-245 of revised manuscript, it reads "To validate the above hypotheses, we designed three mutations, including I464V TM8 , L467I TM8 and V468I TM8 .
The electrophysiological experiments indicate that mutations do not affect glutamate binding. For these three mutants, application of glutamate in the external solution can significantly activate anion current with similar efficacy as that of wild-type hEAAT2 (Fig.  4d). However, these mutations lead to a significant decrease in sensitivity to WAY-213613, with Ki increased to ~0.17 μM, ~0.46 μM and ~0.20 μΜ for I464V TM8 , L467I TM8 and V468I TM8 mutants, respectively (Fig. 4e), supporting our speculations that the I464 TM8 , L464 TM8 and V468 TM8 residues are crucial for the binding specificity of hEAAT2 with WAY-213613.".
We also attached related functional data in this response file for your convenience just as shown in Figure 2*.
3. The cryoEM maps provided show clear density for glutamate and WAY-213613. Interestingly, they also show density for a DDM molecule in the glutamate binding site in the WAY-213613-bound structure. This reviewer feels that this is a very interesting observation that warrants discussion in the manuscript. Could it be that inhibition by WAY-213613 causes other molecules such as detergents -or more physiologically relevant -lipids to interact with residues in the glutamate binding site and also contributing to inhibition of transport?

Reply:
We appreciate reviewer's comment and agree with the reviewer that the lipid molecule may contribute to the inhibition of glutamate transport by WAY-213613 across the membrane. We also briefly discussed this structural observation in the lines 200-207 of revised manuscript as reviewer suggested. It reads "In the cryo-EM map of hEAAT2 W complex, a DDM-like molecule was found to be sandwiched between TM1 and the unwound helix of TM8. Its hydrophobic tail points towards the extracellular side. The head group is positioned proximal to the bromofluorophenol group and form hydrophobic interactions to stabilize the WAY-213613 binding ( Supplementary Fig. 4b and 4e). Therefore, we speculate that a lipid molecule in the membrane environment may occupy the binding position of the DDM and participate in the inhibitory effects of glutamate transport by the WAY-213613.". 4. This manuscript lacks any discussion of the results or a conclusion -these are essential aspects to scientific literature and without them this manuscript is incomplete. This reviewer feels especially strongly about this given that inhibition of glutamate transporters therapeutically is somewhat controversial and warrants, at the very least, further thought and discussion.

Reply:
We appreciate reviewer's comments and we have supplemented a discussion in the lines 235-301 of revised manuscript. It reads "hEAAT2 is the most abundantly expressed excitatory amino acid transporter in astrocytes, which is obligate for the uptake of glutamate from the synaptic cleft into astrocytes to prevent neuronal cytotoxicity 3 . Here, we report the cryo-EM structure of hEAAT2 Glu in complex with glutamate, which reveals the molecular details of how hEAAT2 recognizes the glutamate. By comparison with eukaryotic homologs (hEAAT1 19 , hEAAT3 20 , hASCT2 22 ) and prokaryotic homologs (GltPh 14 , GltTk 17 ) ( Supplementary Fig. 6), the key interaction residues involved in substrate binding are highly conserved between hEAAT2 and the above-mentioned homologs. Meanwhile, we found that a unique residue S441 HP2 is exclusively present in hEAAT2 and it is substituted by the conserved glycine at the corresponding position of other homologs ( Supplementary Fig. 7). Electrophysiological experiments showed that the affinity of S441G HP2 to glutamate can be significantly increased as compared with that of wild-type hEAAT2 (Fig. 2h). Notably, the S441 HP2 is located at the HP2 tip, and does not directly take part in the interaction with glutamate (Fig. 2g). Thus, we speculate that S441 HP2 may affect the rate of transport glutamate. Further experiments will be required to clarify the functional roles of S441 HP2 in hEAAT2.
In this study, we also resolved the structure of hEAAT2 in complex with the inhibitor WAY-213613, which clearly elucidates the binding pocket of hEAAT2 for WAY-213613. The hEAAT2 W complex is stabilized at the inward-facing conformational state. The α-carboxyl and α-amino and the aniline groups of WAY-213613 form contacts with residues S364 HP1 , D475 TM8 and R478 TM8 and share an overlapped binding site with the substrate glutamate, in line with a notion that WAY-213613 is a competitive inhibitor. In the hEAAT2 W complex, the HP2 undergoes a remarkably conformational change and rotates away from the HP1, thus creates enough space for the WAY-213613 binding. Interestingly, the S441 HP2 was determined to form a hydrogen bond with T395 TM7 and consequently stabilize the HP2 in an open conformation, which turns out that this interaction is critical for the WAY-213613 binding ( Fig. 3c and 2f). Meanwhile, the bromofluorophenol group of WAY-213613 is wrapped by some hydrophobic residues from TM8, TM7b and HP2b, such as I464 TM8 , L467 TM8 , V468 TM8 , M450 HP2 and L447 HP2 (Fig. 3c). Based on structural comparison and sequence alignment, the residues I464 TM8 , L467 TM8 and V468 TM8 from TM8 in hEAAT2 are varied as compared with the corresponding residues of other hEAATs (Fig. 4c). The electrophysiological experiments demonstrate that the above three residues are critical for the inhibition of hEAAT2 by WAY-213613 with high potency and mutation in each of three residues substantially reduces the inhibitory efficiency of hEAAT2 by WAY-213613 (Fig. 4e). Considering the I464-L467-V468 cluster in hEAAT2 is substituted by Val-Leu-Ile in hEAAT3 or Ile-Ile-Val in hEAAT1/hEAAT4/hEAAT5, respectively, we speculate that the I464-L467-V468 cluster from TM8 acts as a key structural determinant for the selective inhibition of hEAAT2 by WAY-213613.".

Reply:
We thank reviewer's suggestion and have made the correction as "Glutamate is the predominant excitatory neurotransmitter, which serves as a key role in the development of the mammalian central nervous system, and participates in normal brain function, such as incorporating learning, cognition and memory 1 ." in the lines 32-34 of revised manuscript.
Reply: We appreciate this comment and "the" in the second instance has been removed from the corresponding position in revised manuscript.
• Line 27: It should be noted that excessive concentrations are neurotoxic.

Reply:
We thank reviewer's suggestion and corrected the corresponding description as "Also, excessive glutamate can lead to excitotoxicity, which may kill neuronal cells through excessive stimulation of glutamate receptors 2 ." in the lines 34-36 of revised manuscript.
• Line 32: "Buffering glutamate" doesn't really make sense. Should be replaced with "transporting glutamates back up into the pre-synapse or astrocytes".

Reply:
We thank reviewer's suggestion and have corrected the sentence in the lines 37-41 of revised manuscript. Now it reads "EAATs are known as excitatory amino acid transporters including five subtypes (EAAT1-EAAT5), which are responsible for the removal of glutamate from the synaptic cleft by rapidly binding and transporting glutamates back up into the pre-synapse or astrocytes, which contributes to the termination of synaptic activity and to the clearance of potentially cytotoxic extracellular glutamate 3 .".
• Lines 39-40: As noted above in major comments, more detail is warranted to explain how EAAT2 may be an important therapeutic target.

Reply:
We thank reviewer's comment. We have added more details in the lines 45-48 of revised manuscript to justify hEAAT2 may be an important therapeutic target. It reads "Deficiency of hEAAT2 causes progressive neuronal death, and psychiatric or neurological diseases, including major depressive disorder, epilepsy, Alzheimer's disease, stroke, Parkinson's disease and amyotrophic lateral sclerosis (ALS), and thus hEAAT2 represents a potential therapeutic target 6-8 .".
• Line 41: Ph for GltPh should be in subscript.

Reply:
We appreciate very much for reviewer's suggestions. We have checked throughout the manuscript and all "GltPh" has been corrected as "GltPh" in revised manuscript. Moreover, we have also changed all "GltTk" to "GltTk".
• Line 47: The substrate binding site is described as being "close to HP1 and HP2". There are several substrate-bound structures of glutamate transporters and given that this is a key finding of this paper, what is already known about the substrate binding site should not be glazed over. More detail is required.

Reply:
We thank reviewer's suggestions. We have included more details about substrate binding site in revised manuscript (lines 53-56). It reads "Previous studies have shown that the individual subunit in EAATs can transport the substrate independently 11-13 , and the substrate binding site is constituted by the central unwound region of TM7 (NMDGT motif), TM8 and tips of HP1 and HP2 3,9 .".
• Line 52: What is the percentage identity between human EAAT2 and other human EAATs? How much does it vary in functionally important regions?

Reply:
We appreciate reviewer's comments. We carried out sequence alignment between hEAAT2 and its homologs and hEAAT2 shares 34% sequence identity with GltPh and GltTk, 50% sequence identity with hEAAT1 and hEAAT3, 42% and 39% sequence identity with hASCT1 and hASCT2, respectively. The substrate binding pocket including TM7, TM8, HP1 and HP2 shares high sequence conservation between hEAAT2 and its homologs ( Supplementary Fig. 7). We added sequence identity information in revised manuscript (line 60-63). It reads "However, it is still desirable to determine the structure of hEAAT2, as it shares low sequence identities with these homologs (34% sequence identity with GltPh and GltTk, 50% sequence identity with hEAAT1 and hEAAT3, 42% and 39% sequence identity with hASCT1 and hASCT2, respectively).".
• Line 53: It would be useful at this point of the paper to know the affinities of WAY-213613 for different human EAATs rather than at line 157.

Reply:
We thank reviewer's suggestion. We have introduced the affinity information of WAY-213613 in the lines 63-65 of the revised introduction. It reads "WAY-213613 is a potent and highly selective inhibitor for hEAAT2 (IC50 is 85 nM), which has 59-fold and 44-fold affinity over those of hEAAT1 and hEAAT3 (IC50 is 5 and 3.8 μM, respectively) 26 .".
• Supplementary Figures 2 and 3: It is difficult to see the densities. Please change the colors. Also, the densities for substrates and inhibitors should be shown in these figures.

Reply:
We thank reviewer's comment. We revised the figures for clarification. The revised Supplementary Fig. 2e   • Lines 67-68: References are missing for the structures referred to.
• Line 76: What do the authors mean by "HP1 is attached to the inner membrane"? This reviewer is not familiar with the concept of a protein attaching to the membrane.

Reply:
We thank reviewer's comment. We have corrected the corresponding description as "In our hEAAT2 structures, the HP1 is situated approximately parallel to the membrane plane and almost all exposed in the cytoplasm (Fig. 1d)." in the lines 101-102 of revised manuscript.
• Supplementary Figure 4 and Lines 86-88: While it has been described that CHSresembling densities could be identified, this is not clearly demonstrated in Supplementary Figure 4. The authors should show how CHS fits into the density with more detail if this is their claim.

Reply:
We thank reviewer's comment. We have corrected the depiction of CHS in the lines 112-117 of revised manuscript as "In fact, CHS resembling densities are determined to be located in the cavity formed by TM3, TM6 and TM8 at the inner lobe of plasma membrane ( Supplementary Fig. 4c and 4d ), which was also found in the maps of hEAAT3 35 and GltPh 14 (6S3Q and 6X15, respectively), suggesting that cholesterol may be correlated to the assembly or function of SLC1 transporters and their homologs.".  Fig. 4c and S4d in revised manuscript. c. and d. Density of CHS in hEAAT2 Glu and hEAAT2 W complex, respectively. CHS is colored in yellow and the densities are shown as meshes.
• Supplementary Figure 4: The colors for the hairpins are only shown in one of three protomers. Either say this in the legend or show it in all three protomers. Also, HP2 is listed as being blue when really it looks purple. Furthermore, WAY-213613 is said to be colored in pink, when it is actually red, and this can be very confusing with the pinkmagenta used to color HP2 in this panel. Please use more distinct and consistent colors for these figures. Labels may also be helpful.

Reply:
We thank reviewer's comment and feel sorry for the improper color scheme. We updated Supplementary Fig. 4 and the corresponding figure legends for clarity in revised manuscript.
We also attached the revised Supplementary Fig. 4 here (Figure 9*). • Lines 93-95: This sentence seems out of place and is unnecessary at this point in the manuscript.

Reply:
We thank reviewer's comment. We have removed this sentence from revised manuscript.
• Figure 2: I see beta sheets in the structure, but no beta sheets have been mentioned. Please explain.

Reply:
We thank reviewer's comment. We have added these beta sheets information in the lines 95-97 of revised manuscript. Now it reads "These secondary structures assemble into a scaffold domain (TM1, 2, 4, 5 and two extracellular beta sheets insert in TM4b and TM4c) and a transport domain (TM3, 6-8 and HP1, HP2), respectively.".
• Figure 2: Density for glutamate should be shown in a close-up view.

Reply:
We thank reviewer's comment. We updated Fig. 2 with added a close-up view for glutamate into the panel b for clarity in revised manuscript. Reply ： We thank reviewer's suggestion. We have added the descriptions of the structural comparison between hEAAT2 and eukaryotic homologs (hEAAT1, hEAAT3, hASCT2) and prokaryotic homologs (GltPh, GltTk) in the main text.
In the lines 136-142, it now reads "By comparison with eukaryotic homologs (hEAAT1 19 , hEAAT3 20 , hASCT2 22 ) and prokaryotic homologs (GltPh 14 , GltTk 17 ), key residues involved in substrate binding are highly conserved between hEAAT2 and the above-mentioned homologs ( Supplementary Fig. 6). However, R478 TM8 in the hEAAT2 is substituted by C467 TM8 at the corresponding position in the neutral amino acid transporter hASCT2 ( Supplementary Fig. 6e), which contributes to the substrate selectivity of hASCT2 for neutral amino acids 20 .".
We have also presented a new Supplementary Fig. 6 and we attached it as Figure 4* in this response.
• Line 109-110: "Upon the glutamate binding" should be replaced with "when glutamate is bound" because here, we are talking about the release of substrate.

Reply:
We thank reviewer's comment and have corrected this sentence as reviewer suggested (lines 154-156). It now reads "Compared with inward-facing hEAAT3 at apo state (PDB ID: 6X3F) 36 , we found that the HP2 in the hEAAT2 Glu complex remarkably shifts towards the intracellular side when glutamate is bound (Fig. 2g), which would prevent the escape of substrate.".
• Lines 115-118: Is S441 important for substrate transport? This warrants functional investigation, especially since it is not conserved amongst the different homologues. Why are these interactions of S441 significant?

Reply:
We appreciate reviewer's comment. We have performed electrophysiological experiments to explore the functional roles of the S441 and added a discussion in the lines 165-178 of revised manuscript. It reads "To investigate the functional role of the S441 HP2 , we substituted this residue with glycine (S441G HP2 ) and performed electrophysiological recordings by applications of various concentrations of glutamate. Strikingly, we found that the S441G HP2 mutant displays higher sensitivity to glutamate with the Km at 0.40±0.03 μM (Fig. 2h), which is increased by ~77-fold higher than that of wild-type hEAAT2, although the S441 HP2 is not directly involved in the glutamate binding ( Fig. 2c and 2d). This residue is exclusively present in the hEAAT2, as compared with the conserved glycine at the corresponding position in other homologs ( Supplementary Fig.  7). We speculate that the unique residue S441 HP2 probably provides additional interactions to stabilize the HP2 at a sealed conformation and prohibit glutamate release from the intracellular side. Thus, mutation in the S441 HP2 might rupture these interactions, facilitate the glutamate release, accelerate the glutamate uptake cycle, and consequently biases the hEAAT2 towards a channel with higher open probability. The apparent Ki of WAY-213613 for the mutant S441G HP2 was reduced to 0.26 μM (Fig. 2f), which hints that this site also affects the binding of the inhibitor in a certain way.".
• Lines 124-125: "WAY-213613 has no effect on ionotropic and metabotropic glutamate receptors and thus it's a potential tool for elucidating the function of hEAAT2." We already know the function of EAAT2; this is a vague sentence and does not tell the reader why they should care about understanding how WAY-213613 binds to EAAT2. Careful thought, consideration and explanation is warranted here.

Reply:
We thank reviewer's comment. We agree with the reviewer that this sentence does not clearly show why we study structure of WAY-213613 bound hEAAT2. The WAY-213613 is a potent and highly selective inhibitor for hEAAT2 (IC50 is 85 nM), which has 59-fold and 44-fold affinity over those of hEAAT1 and hEAAT3 (IC50 is 5 and 3.8 μM, respectively). It is desirable to understand how the WAY-213613 specifically bind with hEAAT2.
We include the selectivity information in the lines 63-65 in the revised introduction as the reviewer 1 suggested. It reads "WAY-213613 is a potent and highly selective inhibitor for hEAAT2 (IC50 is 85 nM), which has 59-fold and 44-fold affinity over those of hEAAT1 and hEAAT3 (IC50 is 5 and 3.8 μM, respectively) 26 .".
In the lines 180-181, we modified the previous statement. It now reads "The WAY-213613 displays high selectivity for the hEAAT2, thus it is desirable to investigate its specificity differences between hEAATs.".
• Line 128 and Figure 3B: A more zoomed-in figure of WAY-213613 in the density map should be shown.

Reply:
We thank reviewer's comment. We updated Fig. 3 with added a close-up view for WAY-213613 into the panel b in revised manuscript. I attached the revised Fig. 3b here for your convenience. • Figures 2D and 3D are mirror images of each other. Please amend for ease of interpretation.

Reply:
We thank reviewer's comment. We updated Fig. 2d and Fig. 3d for ease of interpretation in revised manuscript. We attached the original and revised Fig. 2d and Fig.  3d here for your convenience. • In several figures, two structures are overlaid with one structure shown in gray and the other shown in green as detailed by the figure legend. In these figures, HP2 is colored in red, but it is not detailed in the figure legend which structure this red (HP2) belongs to. Please clarify.

Reply:
We thank reviewer's comment. We updated the related Figure legends to clearly identify the attribution of HP2 in revised manuscript.
• Supplementary Figure 6: Please put PDB IDs into figure legends.

Reply:
We thank reviewer's reminder. We have added PDB IDs into the corresponding Figure legends.
• Lines 168-181: There are also structures of TBOA bound to GltPh in outward-facing structures. These should also be discussed; how do they fit into this story?

Reviewer #3 (Remarks to the Author):
EAATS play a critical role in maintaining low glutamate concentrations in the synaptic cleft of the CNS and they are involved in several neurological diseases as well as cancer. Therefore, it is important to understand how these proteins work and how different ligands and inhibitors bind to these transporters. A few structures of the glutamate transporter family have been already published, and the complete transport mechanism of the SLC1A family is well known.
The authors describe 2 new Cryo-EM structures of EAAT2 in the presence of substrate and the inhibitor WAY-213613 and they reveal a new inhibitor binding site for this glutamate transporter family. The manuscript is well written and the methodology used is appropriate.
Although the authors present the structure of the human EAAT2 in the presence of glutamate and the inhibitor WAY-213613, no other experiment was performed to understand and validate the inhibition mechanism by WAY-213613. No mutagenesis analysis or functional analysis was done to corroborate the findings in the Cryo-EM structures. There is no validation in the paper, other than the structures, suggesting that this new inhibitor binding site is real.
This could be done, for example, by mutating key amino acids that interact with the inhibitor WAY-213613 to see if the inhibition could be abolished. This could be analysed either by binding experiments or structural determination. This would be a good way to validate the new inhibitor binding site.

Reply:
We appreciate the reviewer's suggestion about functional experiments. We have performed electrophysiological experiments with the mutants transiently transfected HEK293T to validate residues that coordinate with WAY-213613.
And we have supplemented the related content in revised manuscript. In the lines 237-245, it reads "To validate the above hypotheses, we designed three mutations, including I464V TM8 , L467I TM8 and V468I TM8 . The electrophysiological experiments indicate that mutations do not affect glutamate binding. For these three mutants, application of glutamate in the external solution can significantly activate anion current with similar efficacy as that of wild-type hEAAT2 (Fig. 4d). However, these mutations lead to a significant decrease in sensitivity to WAY-213613, with Ki increased to ~0.17 μM, ~0.46 μM and ~0.20 μΜ for I464V TM8 , L467I TM8 and V468I TM8 mutants, respectively (Fig. 4e), supporting our speculations that the I464 TM8 , L464 TM8 and V468 TM8 residues are crucial for the binding specificity of hEAAT2 with WAY-213613.".
We also attached the related functional data in this response file (see Figure 2*).
I believe the validation reports are mixed up at my end. In the validation report that seems to correspond to the EAAT2-WAY213613, the structures of the ligands Y01 have issues in bond lengths and bond angles. As the paper's main outcome is a new inhibitor binding site of the EAATs, it would be good to see omit maps for the ligands to better appreciate the densities around them.

Reply:
We thank the reviewer for pointing out this issue. We have refined the model. The bond lengths and bond angles of the Y01 molecule look good now and no outliers reported in the new validation reports. By the way, the Y01 is the CHS molecule, instead of the inhibitor WAY-213613.
For these cryo-EM structures, we could not generate omit maps for the ligand similarly to the routinely prepared ones using structures determined via X-ray crystallography. Moreover, both of our structures are bound with ligand glutamate or WAY-213613 and their binding site are overlapped, and thus we cannot prepare a difference map for either.
Another aspect that could be investigated is the difference between monomers. Have the authors observed the same density for the WAY-213613 in the three monomers forming the trimer? A symmetry expansion and focused refinement could be performed to identify any structural variation between protomers.

Reply:
We appreciate reviewer's comment. The WAY-213613 was present during the whole course of sample preparation, including protein expression at the final concentration of 5 μM for 60 h, protein purification at the final concentration of 5 μM and also grids preparation at the final concentration of 200 μM. Considering the high binding affinity of WAY-213613 to hEAAT2 (Ki ~80 nM), we believe that WAY-213613 is most likely to bind with all of three subunits. In our hEAAT2 W map, the WAY-213613 density in three subunits is nearly identical (Figure 14*), further supporting that hEAAT2 is fully occupied by the WAY-213613.  Fig. 7 in revised manuscript. a-c. Currents recorded when 1000 μM glutamate are applied to the hEAAT2 mutants (D475A TM8 and R478A TM8 ) or wild-type hEAAT2expressed HEK293T cells. d. Current traces obtained when glutamate was applied at the indicated concentrations to the hEAAT2 mutant S441G HP2 -expressed HEK293T cells. e-g. Current traces obtained when WAY-213613 was applied at the indicated concentrations to hEAAT2 mutants (D475A TM8 , R478A TM8 and S441G HP2 )-expressed HEK293T cells. All experiments were executed at 0 mV and the experimental procedure was the same as the one described in the method. Fig. 9 in revised manuscript. a-c. Current traces obtained when glutamate was applied at the indicated concentrations to the hEAAT2 mutants (I464V TM8 , L467I TM8 and V468I TM8 )-expressed HEK293T cells. d-f. Current traces obtained when WAY-213613 was applied at the indicated concentrations to hEAAT2 mutants (I464V TM8 , L467I TM8 and V468I TM8 )-expressed HEK293T cells. All experiments were executed at 0 mV and the experimental procedure was the same as the one described in the method.

Figure 2*. New Supplementary
2. Could the authors provide more details about electrophysiology? For example, holding potential.